In responsive cells, type I and type II interferons induce 15-20 different proteins that are thought to mediate the host of cellular regulatory effects such as changes in cell structure, activity, differentiation, proliferative capacity, presentation of major histocompatibility antigens, and anti-viral activity. However, the functions for few of these proteins have been ascribed to date, although such knowledge is fundamental to understanding and exploiting the potential of the interferons as therapeutic agents. One such interferon-induced protein of unknown function has a molecular weight of 15 kDa. This protein must be important for the ability of cells to mount an interferon response since it is rapidly induced and its accumulation parallels the appearance of antiviral activity. We have previously proposed that this polypeptide, termed Ubiquitin Cross-Reactive Protein (UCRP) because of its immunological and sequence similarity to ubiquitin, represents a function-specific homolog of ubiquitin. More recently we have shown that, like ubiquitin, UCRP is subject to both constitutive and interferon-induced covalent conjugation to a spectrum of intracellular proteins. The long-term objectives of this proposal are to understand the role of UCRP conjugation in normal cellular regulation and in response to interferon. The specific aims of the proposal are: (1) to physically characterize recombinant UCRP stability by CD measurements and to crystallize the protein for subsequent structure determination; (2) to examine the dynamics of intracellular pools of free and conjugated UCRP in normal and interferon-induced cells using affinity-purified polyclonal antibodies against UCRP in conjunction with pulse-chase studies and inhibition of UCRP induction with specific antisense oligonucleotide probes; (3) to study the conjugation of UCRP to cytoskeletal proteins using the anti-UCRP antibodies as immunohistochemical probes for localization of the adducts; (4) to characterize the enzymological steps in UCRP conjugation and isolate by affinity and FPLC methods the enzyme(s) responsible for this unique post-translational modification; and (5) to clone, sequence, and expressed recombinant UCRP conjugating enzyme(s) using current molecular biological techniques. The resulting characteristics of the UCRP conjugating system will be compared to the parallel but distinct ubiquitin ligation pathway.